Normal tissue toxicity to the gastrointestinal (Gl) tract occurs frequently with chemoradiotherapy and represents a major clinical challenge. Unfortunately, no effective treatments exist to combat this significant clinical problem. The luminal epithelia of the Gl tract exist in physiologic hypoxia, and thus hypoxic signaling is crucial to the their survival and normal functions. Hypoxia-inducible factor-1 (HlF-1) is a transcription factor at the heart of the cellular hypoxia response whose levels fluctuate inversely with cellular oxygen tension. Stabilized HIF-1 expression has been shown to be critical for normal intestinal homeostasis as well as survival during inflammatory challenges. HIF-1 is required to maintain several basic functions of the intestine, such as barrier and absorptive functions. Augmenting HIF-1 expression in the intestine improves epithelial barrier functions, increases nutrient absorption and reduces apoptosis in response to infection and inflammatory stress. Conversely, intestinal specific knockouts of HIF-1 cause increased epithelial apoptosis in murine models of colitis. The oxygen dependent regulation of HIF is chiefly mediated through the prolyl hydroxylase domain (PHD)-containing proteins which hydroxylate proline moieties on HIF, and serve as a recognition site for the von Hippel Lindau (VHL) protein, which targets HIF for proteasomal destruction. To date, three oxygen-dependent prolyl hydroxylases have been identified (PHD1-3), however, the roles of each isoform in intestinal homeostasis is not clear. Because PHD proteins regulate HIF levels, and HIF, in turn protects the gut from inflammatory stress, it is our hypothesis that the PHD proteins regulates radiosensitivity in the gastrointestinal tract, such that deletion of specific PHD isoforms will afford radioprotection of the gut through HIF-medlated effects on epithelial integrity and crypt regeneration. In this proposal, we will: 1) investigate the specific contributions of PHD 1-3 on radioprotecting the Gl tract from hypofractionated and fractionated radiation with Project 2 and Core B, 2) determine the mechanisms of radioprotection by PHD inhibition with Project 3; 3) explore the role of PHD 1-3 in the radiation response ofthe intestinal stem cell with Project 2, 4) determine the efficacy of a small molecule PHD inhibitor to protect the Gl tract with Project 2; and 5) determine the therapeutic efficacy of PHD 1-3 knockouts and small molecule inhibitors of PHD activity on tumor growth in response to radiation relative to normal tissue radioprotection with Project 4. This project represents a new paradigm change in the development of radioprotectors in that we are investigating the physiological basis of tissue radioprotection through alterations in epithelial barrier function.